Method for detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations

ABSTRACT

The invention relates to a method for detecting specific target-cells in a simple and time saving way, using paramagnetic particles, antibodies recognizing the Fc portions of target-cell associating antibodies and target-cell associating antibodies directed to specific antigen determinants in the target-cell membranes. Incubation of the cell suspension with a mild detergent and/or second set of antibodies or antibody fragments, prelabeled or not with fluorescent agents, metallocolloids, radioisotopes, biotincomplexes or certain enzymes allowing visualization, with dramatically increase the specificity of the method. The method can further be used for isolation of the target-cells by magnetic field application and kit for performing the method according to the invention is described.

The present invention relates to an immunomagnetic method for detectionof specific target cells in cell populations and solutions of cellpopulations. The invention also relates to a kit for performing themethod in different cell populations.

In biology, biochemistry and adjacent fields it is considerable need formethods in which chemical entities are linked together. Such methodshave an increasing importance in research regarding both normal andabnormal cell populations. Especially when solid supports are used cellscan be immobilized, detected and isolated and purified. Furthermore, thecell content may be examined using a range of sofisticated methods. Itis also of importance to be able to isolate the cells in viable forms.

Affinity binding is a sofisticated way of linking chemical/bio-chemicalentities together. In such a method a pair of binding partners, whichfor example are attached to the substances to be linked, bind to eachother when brought in contact. One of the binding partners in such alinkage may be represented by a molecule on the cell surface. Severalsuch binding partner systems are known, such as antigen- antibody,enzyme- receptor, ligand- receptor interactions on cells and biotin-avidin binding, of which antigen-antibody binding is most frequentlyused. A hapten/anti-hapten binding pair method has also recently beensuggested WO 91/01368.

When such methods are used for isolation of specific cells, which arethe subject for further various examinations, it is necessary to reversethe linkage without producing destructive effects on the bindingpartners, which ideally should recover their function upon returning tothe original conditions. This is not always the case, although it isproposed a method for adequately cleaving antigen/anti-antigen andhapten/anti-hapten linkages (PCT/EP91/00671, PCT/EP90/01171).

Methods are known in which one of the binding partners is attached to aninsoluble support, such as paramagnetic particles, and by whichisolation of target cells in a mixed cell population is performed asnegative isolation or positive isolation. In a negative isolationprocedure the unwanted cells can be removed from the cell preparation byincubating the cells with antibody-coated particles, specific for theunwanted cells. Following the incubation thecell/antibody/particle-complex can be removed using the particles,leaving the wanted target cells behind. This result is often notsatisfactory, since the wanted cells are left in the cell population,more or less purified, and also since the intention of the isolationprocedure is to examine and/or perform further studies on the specifictarget cells. Attempts have been made to use particles for positiveisolation, in which the wanted target cells are removed from the mixedcell population. These procedures have, however, been directed tocertain target cells and are not suited for all target cell systems. Apositive isolation procedure involving the hapten/anti-hapten linkagesystem has recently been proposed (PCT/EP90/01171) and also a method forisolating haemopoietic progenitor cells from bone marrow(PCT/EP90/02327). The latter is directed to use a combination ofpositive and negative selection for the purpose of isolating andpossibly growing specific cells, i.e. haematopoietic progenitor cells,in the bone marrow, and is dependent upon removal of the particles.

PCT/EP90/01171 relates to a method of connecting target cells to aninsoluble support by using the abilities of hapten, anti-haptenantibodies and anti-cell antibodies to bind to each other, thusconstructing a linkage between the insoluble support, i.e. particle, andthe target cell, consisting at least of hapten and anti-hapten antibodyor combinations of hapten and anti-hapten antibodies andanti-anti-hapten antibodies or secondary anti-cell antibodies. The latercleavage of the complex is performed by again exposing it to hapten orhapten analogue. Thus the constructed link always consists of hapten inaddition to 1 or more elements. The method is directed to unspecifiedtarget cells and is directed to isolation of target cells and release ofthe insoluble support.

There is a need for a simple linkage to connect a target cell to aninsoluble support, which do not involve compounds of a ratherunspecified haptene-group, and which is directed to detection ofspecific target cells, with a minimum of unspecific cell association andwhich render unnecessary a later cleavage between the insoluble supportand the specific target cell.

Thus the object of the present invention is to detect for diagnosticpurposes specific target cells when used in a blood and bone marrow,without the problem with unspecific binding to normal cells. Itrepresents a sensitive detection method for a variety of cell types,such that a high number of cells can be readily screened in themicroscope and the procedure is rapid and simple. Furthermore, thepresent method can be used for isolation of cells for biochemical,biological and immunological examination, and for studying of specificgenes at the nucleotide or protein level, in addition to culturing thecells, without the need for cleaving the cell-particles complex. Afurther object of the invention is to provide a kit for performing themethod as characterized in the claims.

The intensions of the inventions are obtained by the method and kitcharacterized in the enclosed claims.

The method for immunomagnetic detection of target cells in a mixed cellpopulation and physiological solutions containing cell populations issuitable for detection, but may also be used in positive isolation ofspecific types of both normal cells and patogenic cells. The methodcreates a linkage between a specific target cell and an insolublesupport, such as paramagnetic particles , which consists of one or twoelements. The particle is either coated with an anti-cell antibody ofmurine or human origin, directed to the specific antigen determinants inthe membranes of the wanted target-cells, or the particles are coatedwith a polyclonal anti-mouse or anti-human antibody capable of bindingto the Fc-portions of the specific anti-cell antibody directed to theantigen determinants in the target-cell membranes. Instead of using thepolyclonal anti-mouse/anti-human antibody for coating the particles, amonoclonal rat anti-mouse/anti-human antibody may be used. This lastantibody, due partly to its monoclonal origin, may provide a morespecific binding to the anti-cell antibody and reduce the risk forpossible cross-reactions with other cells in solutions, such as blood.Furthermore, incubation of the cell suspension with a mild detergentand/or second set of antibodies or antibody fragments, prelabeled or notwith fluorescent agents, metallocolloids, radioisotopes,biotin-complexes or certain enzymes allowing visualization, willdramatically increase the specificity of the method.

In the following a more detailed disclosure of the method is presented,using cancer cells as the target-cells for detection and possibleisolation. The method is, however, not limited to cancer cells and thedisclosure shall not limit the method to this particular field of use,since the method is suitable within a range of cytological researchareas.

In the management of cancer patients, the staging of the disease withregards to whether it is localized or if metastatic spread has occurredto other tissues, is of utmost importance for the choice of therapeuticalternative for the individual patient. Malignant cells spread by directinvasion into the surrounding tissue, through the lymphatics or by thedistribution of tumor cells in the blood to distant organs, includingthe bone marrow and the central nervous system and the cerebrospinalfluid.

Detection of metastatic tumor cells has, until recently, relied onmorphological methods using light and electron microscopy on biopsiedtumor specimens, on smears of bone marrow and peripheral blood, and onslides prepared after cytosentrifugation of various body fluids. Sincethe advent of monoclonal antibodies recognising antigens predominantlyexpressed on the surface of different types of malignant cells, theidentification of metastatic cells has, to an increasing extent, alsoinvolved immunocytochemistry and immunofluorescence. Thus, slidesprepared from biopsied tumors or cytosentrifugates have been treatedwith monoclonal antibodies, and the binding of these to the tumor cellsis visualized calorimetrically or by fluorescence. The latter methodrequires the use of a fluorescence microscope, alternatively preparing acellsuspension an use a flow cytometer.

The previous methods suffer from limited sensitivity and/or specificity,and is usually laborious and time consuming, also requiring a highdegree of expertise. Flow cytometric examiniations also involveexpensive equipment.

The morphological methods for the detection of tumor cells in blood andbone marrow are much less sensitive than methods involvingimmunocytochemistry and immunofluorescence (Beiske et al., Am. J.Pathology 141 (3), September 1992). Also the latter methods are,however, inadequate in cases where the tumor cells represent less than1% of the total number of nucleated cells. Flow cytometry may providebetter sensitivity than the methods involving the use of a microscope,but requires the availability of a high number of cells, and alsoinvolves several technical difficulties. Thus, aggregation of cells maycause problems, and the method does not provide possibilities todistinguish between labeled tumor cells and unspecifically fluorescingnormal cells.

The invention allows for a very sensitive detection of, for example,metastatic tumor cells, since a high number of cells can readily bescreened in the microscope and the attached magnetic beads are easilyrecognisable. The monoclonal antibodies used bind with sufficientspecificity to, for example, tumor cells and not to other cells than thetarget-cells present in mixed cell suspensions, like blood, bone marrow,and in other tumor manifestations, such that all cells with attachedbeads represent the target-cells. In addition, the procedure is rapidand simple, and can be performed by any investigator with access to aconventional microscope.

The novel method involves the binding of monoclonal antibodies, e.g. ofmurine or human origin, that specifically recognize antigens present ontumor cells, and not on the normal cells in question, or for otherpurposes to specified subpopulations of normal cells, to paramagneticparticles, either directly or to beads first covered with antibodiesspecifically recognizing the respective antibodies, or the Fc-portion ofIgG antibodies, that bind to the tumor cells. The cell bindingantibodies may be of the IgG or IgM type or being a fragment of ab IgGor IgM. Examples of used anti-target-cell antibodies may be thosedirected against groups of antigen determinants, for example CD56/NCAMantigen (MOC-1), Cluster 2 epithelial antigen (MOC-31), Cluster 2 (MW˜40kD) antigen (NrLu10) (Myklebust et al. Br. J. Cancer Suppl. 63, 49-53,1991), HMW-melanoma-associated antigen (9.2, 27) (Morgan et al.,Hybridoma, 1, 27-36, 1981), 80 kD, Sarcoma-associated antigen (TP1 &TP3) (Cancer Res. 48, 5302-5309, 1988), mucin antigens (Diel et al.,Breast Cancer Res. Treatm., 1991), or EGF-receptor antigen (425.3)(Merck), in addition to the anti-pan-human antibody (Bruland et al.,unpublished), which is suitable for detecting human cells among animalcells. The 425.3 antibody is directed towards antigens in both normaland malignant cells. Antibodies can furthermore be directed againstgrowth factor receptors, for example EGF-receptor, PDGF (A and B)receptor, insuline receptor, insuline-like receptor, transferrinreceptor, NGF and FGF receptors, group of integrins, other adhesionmembrane molecules and MDR proteins in both normal cells and abnormalcells, and antigens present on subpopulations of normal cells, inaddition to oncogenic products, expressed on the membranes of normal andmalignant cells and on malignant cells alone, for example Neu/erbB2/HER2. As for the malignant cells, these may be breast, ovarian andlung carcinoma cells, melanoma, sarcoma, glioblastoma, cancer cells ofthe gastrointestinal tract and the reticuloendothelial system, or thetarget-cells may be associated with non-neoplastic diseases, such ascardiovascular neurological, pulmonary, autoimmune, gastrointestinal,genitourinary, reticuloendothelial and other disorders. Furthermore, themalignant cell population may be located in bone marrow, peripheralblood, come from pleural and peritoneal effusions and other body fluidcompartments, such as urine, cerebrospinal fluid, semen, lymph or fromsolid tumors in normal tissues and organs, for example liver, lymphnodes, spleen, lung, pancreas, bone tissue, the central nervous system,prostatic gland, skin and mucous membranes. A more complete list of theantigen determinants and the corresponding antibodies or antibodyfragments used in the present improved method is presented in Table 1.

The method comprises attachment of the antibodies directly to theparamagnetic particles, or the attachment can take place by attachmentto surface-bound antibodies, such as polyclonal anti-mouse antibodies,monoclonal rat anti-mouse antibodies or monoclonal anti-humanantibodies, specifically recognizing the Fc-portion of the saidindividual antibodies. The antibody-coated paramagnetic beads are thenmixed with the suspension of cells to be examined and incubated for 5-10min to 2 h, preferably for 30 min at 0-25° C., preferably at 4° C.,under gentle rotation. The present method may also be performed in achanged order of steps, in that the free target-cell antibodies areadded to the cell suspension, incubated for 5-10 min to 2h, preferably30 min, at 0-20° C., preferably 40° C., under gentle rotation. Theparamagnetic particles, precoated with anti-mouse or anti-humanantibodies are then added to the incubated cell suspension, as describedabove, and the resulting suspension subjected to a further incubation of5-10 min to 2h, preferably 30 min, at 0-25° C., preferably 4° C. undergentle agitation.

Samples of the cell suspension are then transferred to a cell countingdevice, and the fraction of cells with attached beads relative to thetotal number of cells is determined under light microscopy. The numberof antibody-coated beads added to the cell suspension should be between0.5-10 times the number of target cells. When this number is unknown,the amount of coated beads added should be 1-10% of the total number ofcells.

For specific purposes, and in the cases where the density of thetarget-cells is low, for example malignant cells, or the target-cellsrepresent a very low fraction of the total number of cells (≦1%), thetarget cells can be positively separated from non-target cells in amagnetic field. The isolated target cells, can then be enumeratedmicroscopically and the fraction of target cells relative to the totalnumber of cells in the initial cell suspension can be calculated.Moreover, the target-cells may be characterized for the presence ofspecific biochemical and biological features of particular importancewill be the use of such cells for studies in molecular biology. Incontrast to the above cited methods of the prior art, the present methodallows studies and growth of the target-cells without performing acleavage of the paramagnetic particle-target cell linkage. For severalpurposes it is of interest to examine specific genes in a purepopulation of target cells at the DNA, mRNA and protein level, both intumor biopsies as well as in tumor cells present in blood, bone marrowand other body fluids, for example urine, cerebrospinal fluid, semen,lymph, or from otherwise normal tissues and organs, for example liver,lymph nodes, spleen, lung, pancreas, bone tissues, central nervoussystem, prostatic gland, skin and mucous membranes, and in other areasof cytological research activity.

With the methods of prior art, signals obtained on Southern, Northernand Western blots represent the normal cells as well as the tumor cellsin the biopsy. If a single cell suspension is first prepared from thetumor material, and the tumor cells are then positivelyimmunomagnetically detected and separated, any gene studies performed onthis material would represent the target-cells only. This also relatesto for example malignant cells present in mammalian tissues, for examplein bone marrow, peripheral blood, pleural and peritoneal effusions, andother body fluids, for example urine, cerebrospinal fluid, semen andlymph. Studies involving polymerase chain reaction (PCR) methodologywill also gain in specificity and reliability when performed on puretumor cell populations obtained by the new method.

The application of the new method steps may differ depending on type oftissues to be examined.

a) Tissue from solid or needle tumor biopsies is prepared mechanicallyor with mild enzymatic treatment into a single cell suspension, to whichthe primary, specific antibodies or antibody fragments are addeddirectly or after washing the cell suspension with phosphate bufferedsaline or culture medium with or without serum, such as fetal calfserum, bovine, horse, pig, goat or human serum.

b) If the material is a sample of pleural or ascitic effusion,cerebrospinal fluid, urine, lymph or body fluids such as effusions inthe joints of patients with various forms of arthritis, the specificantibodies or antibody fragments are either added to the samplesdirectly, or after centrifugation with or without washings before orafter the cells in the samples are spun down and brought back intosuspension.

c) If the material consists of blood or bone marrow aspirate, themononuclear cell fraction is isolated by gradient sentrifugation on e.g.Lymphoprep before washing, resuspension, and addition of the appropriateantibodies or antibody fragments.

The procedure conditions for a) and b) are established, as exemplifiedby results obtained in successful experiments as those described below.

For c) the results have been found to be influenced by a high number offactors which have been examined in detail. Among these are antibodyconcentration, the ratio of the number of paramagnetic particles versusnumber of cells, incubation times and volumes, type of incubationmedium, and the pH level. The particle to mononuclear cell ratio in allexperiments should be in the range of 0.5/1-2/1, depending on thebinding affinity of the primary specific antibodies or fragments.

A major problem has been unspecific attachment to normal blood or bonemarrow cells of particles coated with either sheep or rat anti-mouseantibodies alone, or in addition with the specific antibodies.Experiments have shown that the unspecific binding is equally highwithout the presence of the specific antibodies, indicating that theproblem is not caused by cross-reactivity of the targeting antibodies tonormal cells. The possibility that the less than optimal specificitycould be caused by ionic binding has been ruled out. Another possibilitywas that subpopulations of normal cells of the B-lineage might adhere tothe particle-antibody complexes. However, immunomagnetic removal ofB-cells from the cell suspension before adding the specificantibodies/antibody-particle complexes did not improve the specificityof the latter.

The problem with the procedure used on is isolated mononuclear fractionsof bone marrow and peripheral blood that some non-target cells mightalso bind paramagnetic particles, has been circumvented or overcome.Thus with sheep-anti-mouse antibody coated particles alone or withspecific antibodies the number of particles unspecifically attached to alow fraction mononuclear blood or bone marrow cells was reduced from anaverage of 10 to about 1 and in paralell the fraction of normal cellswith particles decreased from 1-2% to 0.5-1% or less.

Evidence has been obtained that the problem may be caused by hydrophobicforces associated with the antibodies bound to the paramagneticparticles. Methods for reducing this hydrophobicity is thus claimed. Onesuch method is preincubation of the antibody-coated particles and thecell suspension with mild detergents in suitable concentrations, forexample Tween 20 in concentrations of less than 0.1% for 30 minutes at4° C. When possible selection of the target cells is warranted, the cellsuspension should contain a low concentration of the detergent, e.g.0.01% of Tween 20. In several experiments this procedure has almosteliminated or dramatically reduced the problem of unspecific bindingseen with the mononuclear cell fractions from blood or bone marrow.

The other improvement which, if found warranted, may be used togetherwith the detergent step as follows:

After incubation of the cell suspension with the primary antibodies orantibody fragments and the antibody-coated paramagnetic particles asdescribed in previously, the cell suspension is incubated with a secondset of antibodies or antibody fragments directed against otherextracellular or against intracellular determinants of the target cells,with or without pretreatment with cell fixatives such as formaldehyde oralcohols. These antibodies or their fragments should have beenprelabeled by fluorescent agents, metallocolloids, radioisotopes,biotin-complexes or enzymes like peroxidase and alkaline phosphatase,allowing visualization by per se known methods in the microscope and/ora suitable counting device.

The target cells will both be visualized with the latter method and havebound particles to their surface, and can thus be enumerated.

To simplify the distinction between non-target and target cells, thecell suspension can before the second visualization step either besubjected to cytospin centrifugation or portions of the suspension areattached to caoted glass slides on which the particle-bound cells willbe spread out in a thin layer, facilitating the recognition of thedouble-“stained” cells.

For use in the new procedure, kits will contain for example precoatedparamagnetic particles prepared for each monoclonal antibody. In anotherembodiment the kits contain paramagnetic particles pre-coated with IgGisotype specific anti-mouse or anti-human antibody as one part of it,and different target cell-associated, for example tumor cell, antibodiesas another part. In a third embodiment the kit contains paramagneticparticles precoated with specific anti-Fc antibodies, such as polyclonalanti-mouse, or monoclonal rat anti-mouse, or anti-mouse, or anti-humanantibodies, capable of binding to the Fc-portion the target-cellassociating antibodies, bound to specific anti-target-cell antibodies.In a further embodiment the kit contains other specific antibodies orantibody fragments directed against antigens/receptors within or on thewanted target-cells, where said antibodies or antibody fragments areconjugated to peroxidase, alkaline phosphatase, or other enzymes,together with relevant substrates to such enzymes, or where saidantibody or antibody fragment is bound to non-paramagnetic particleswith specific colours or with bound enzymes such as peroxidase andalkaline phosphatase.

The present method will in the following be illustrated by modelexperiments, examples of the usefulness of the new method and examplesof practical applications. These examples shall not be regarded as inany way limiting the invention.

MODEL EXPERIMENTS

1. Binding of antibody-bead complexes to tumor cell lines with the newprocedure:

To determine antibody concentrations and optimal conditions for thebinding of antibody-paramagnetic particle complexes to tumor cells, alarge panel of cancer cell lines was used. The paramagnetic beads werebound to the cells, either by coating the specific antibodies tosheep-anti-mouse antibody (SAM)-coated paramagnetic particles, or byfirst incubating the cells with the specific antibodies, washing,followed by a second incubation with SAM-coated particles. The resultsof these experiments are given in Tables 2a and 2b, in which + indicatesbinding of several beads to all cells, (+) indicates either a lowernumber of beads bound to each cell, or that not all the tumor cells hadbeads attached to their surface, whereas − reflects no binding, and (−)indicates very weak binding.

2. For detection of tumor cells in the mononuclear fraction of bonemarrow or peripheral blood, model experiments were performed wherespecific antibodies and SAM-coated paramagnetic particles were addedeither to such mononuclear cells or to a cell suspension where adifferent number of cancer cells from in vitro cultivated cell lineswere added to said mononuclear cells. In some experiments, either themononuclear cells, or the malignant cells were prestained with afluorescent dye, to be able to distinguish beteween the two types ofcells. In all experiments, non-binding primary antibodies, and/orsheep-anti-mouse antibody-coated beads were used separately as controls.

TABLE 2a Cell lines Antibodies MCF-7 SKBR3 T47D MDA231 MDA435 DU145FMEX-1 LOX NrLu10 IgG2b + + (+) (+) + Moc31 IgG1 + + + (+) (+) + Moc1IgG1 (+) (+) + 12H12 IgG1 + + + + 2E11 IgG3 + + + + + 5A6 IgG1 (+) + 5F2IgM (+) CC3 IgG2a − − − − CC1 IgM − (+) CU18 IgG1 − − − CU46 IgG1 (+) −− 7F11 IgG1 − − + − − − D7 IgG3 (+) E4SF IgG1? + + (−) − 50%+ 425-3 +− + 9.2.27 + + MUC18 − − − − 2g12 IgG1 + 4b7 IgG1 + BM2 (=2F11) BM7(=7F11) TP-3 TP-1 CEA GINTES IgG 3C9 IgM HH8 IgM 5F4 IgM 3F1 IgG1

TABLE 2b Cell lines Antibodies PM1 MA-11 CRL1435 CRL1740 H-146 Colo205786-0 WIDR NrLu10 IgG2b + + + + + + − Moc31 IgG1 + + + + + + + + Moc1IgG1 + − 12H12 IgG1 + + (+) − − − 2E11 IgG3 (+) + − + − − − 5A6 IgG1 + +5F2 IgM CC3 IgG2a − − CC1 IgM (+) − CU18 IgG1 − − CU46 IgG1 − − 7F11IgG1 (+) + − − − − ID7 IgG3 − − E4SF IgG1? + + + + − − − 425-3 9.2.27MUC18 − 2g12 IgG1 − − 4b7 IgG1 − − BM2 (=2F11) + + BM7 (=7F11) + TP-3TP-1 CEA GINTES IgG + − 3C9 IgM − − HH8 IgM − − 5F4 IgM − − 3F1 IgG1 − −

In several experiments some unspecific binding to the mononuclear cellswas observed, which was found to be unrelated to the nature of thespecific antibody, and which was equally pronounced with SAM-coatedparticles alone. The magnitude of this unspecific binding varied fromalmost 0 to a level between 0.5-2%. This unspecific binding was almosteliminated by mild treatment with detergent, (Tween 20) performed toreduce the problem of hydrophobic cell interactions.

EXAMPLES OF THE USEFULNESS OF THE NEW PROCEDURE 1. Detection ofMicrometastic Neoplastic Disease in Blood and Marrow

Early and reliable diagnosis of spread of cancer cells to blood and/orbone marrow has become increasingly important for the choice of optimaltherapy, possibly curative in many types of cancer, includingcarcinomas, as described in application Example 1. Similar proceduresfor malignant melanoma, sarcoma, neuroblastoma and several other cancershave been established or are under development.

2. Detection of Malignant Cells in Pleural or Ascitic Effusions, and inUrine

The nature of such effusions may represent an important diagnosticproblem, particularly when a low number of cancer cells are presenttogether with normal reactive or epithelial cells. In several cases adefinite diagnosis has been rapidly made with the new method, in caseswhere conventional cytological examination has been negative orinconclusive. A similar advantage can be found in cases of cancer in thekidneys or in the urinary tract and bladder.

3. Detection of Neoplastic Cells in the Cerebrospinal Fluid

As the systemic treatment of many cancer types have improved, thefrequency of cases with symptom-giving brain metastases havesignificantly increased, and in parallell with this, the necessity forearly detection of such spread. With the use of the new procedure even alow number of malignant cells can easily be identified, permittingintervention with therapeutic alternatives at an early stage ofintracranial tumor manifestations.

4. Diagnosis of Cancer in Biopsied Tissue

When cancer is suspected, and tissue biopsies are obtained by surgicalprocedures or by e.g. needle biopsies, a much more simple and rapiddiagnosis can be made with the new method, used on prepared cellsuspensions, compared to conventional morphological or immunohisto- orcytochemical procedures.

Distinction between several alternative cancers can be made by the useof the appropriate antibodies.

5. Identification of Prognostic Indicators

Since the expression of several membrane molecules have been shown tocorrelate with progression of the malignant disease in several cancers,the present method can be used to identify prognostic indicators, forexample as described in application Example 2.

6. Identification of Cells Indicative of Specific Diseases or of DiseaseProgression or State

In various types of rheumatoid diseases (such as rheumatoid arthritis),as well as in allergic, autoimmune, and cardiovascular diseases,identification of the systemic or local presence of specificsubpopulations of cells is important for diagnosis and for determiningthe stage of the disease. Rapid detection of such cell populations withthe new method is therefore of considerable diagnostic and therapeuticimportance.

7. Detection of Subpopulations of Normal Cells

For several purposes, it will be important to detect the fraction of aparticular subpopulation of normal cells in a population. This appliese.g. to liver biopsies where the identification of cells expressing thebiliar epithelial antigen, may be of importance. Similarly, theidentification, and possible isolation of specific endothelial cellsfrom a cell suspension prepared from various normal tissues may bewarranted.

Several of the cell membrane molecules mentioned in sections 1-6 mayalso be used as targets for immunotherapy with several types ofactiviated killer cells or e.g. with immunotoxins. The identificationwith the new method of expression of such molecules is, therefore, alsoof value for determining in which cases such types of therapy should beused.

EXAMPLES OF A PRACTICAL APPLICATION OF THE METHOD Example 1

To diagnose spread of cancer cells in blood and/or bone marrow at anearly stage, we have used in the new procedure the MOC-31, NrLu10, BM2,BM7, 12H12, and MLuC1 anti-carcinoma antibodies to determine whether ornot micrometastatic disease from breast, lung, colorectal, and prostatecancer might be sensitively identified in such body fluids. Thesuccessful results with these antibodies have significant clinicalimplications.

Example 2

The expression of serveral cell membrane molecules have been shown tocorrelate with progression of the malignant disease in several types ofcancer. The detection of binding of such antibodies to respectiveantibodies can therefore be used to obtain information of highprognostic value. Among such antigens are a high number of adhesionmolecules, carbohydrate antigens, glycolipids, growth factor receptorsand carcinoma markers listed below. We have, with the new procedureidentified the binding of particle-antibody complexes to CD44-variants,E-cadherin, Le^(γ), CEA, EGF-r, transferrin receptor, MUC-1 epitope,LUBCRU-G7 epitope, prostate cancer antigen, UJ13A epitope,β₂-microglobulin, HLA-antigens, and apoptosis receptor.

Example 3

Two litres of pleural diffusion from a patient supposed to suffer frommalignant melanoma was obtained. After centrifugation, the cells weresuspended in a volume of 2 ml of RPMI with a 10% fetal calf serum,incubated with 9.2.27 anti-melanoma antibody (10 μg/ml) at 4° C. for 30min, washed and again incubated with Dynabeads SAM M450/IgG2A at 4° C.for 30 min. The cell suspension was then examined under a microscope fordetermining the fraction of cells with paramagnetic cells attached totheir surface. The diagnosis of malignant melanoma was confirmed, asabout 10% of the cells had a significant number of particles rosettes.

Example 4

Biopsied tissue was obtained from a subcutaneous tumor in a case withclinical indications of either small cell lung cancer or a malignantmelanoma. A single cell suspension was prepared from the biopsy, dividedin 2 fractions, one incubated with the 9.2.27 anti-melanoma antibody,and the other with MOC-31 anti-carcinoma antibody (both at 10 μg/ml).The incubation was similar to that used in the example above. None ofthe cells incubated with the melanoma antibody bound any beads, whereasall tumor cells incubated with MOC-31 were positive.

Example 5

Biopsied tissue from a patient suspected to have malignant melanoma wasexamined by preparing single cell suspension, incubating with 9.2.27anti-melanoma antibody, and then following the procedure as above. Mostof the cells were positive with a high number of particle-rosettesattached to their membranes.

Example 6

A pleural effusion from a breast cancer patient was studied to examinewhether tumor cells could be detected in the fluid. One litre of thefluid was centrifuged, the cells resuspended, and in separate vialsincubate with each of 3 different anti-carcinoma antibodies (MOC-31,2E11, 12H12). After completing the procedure as in the previous example,it was found that most of the cells bound to antibody-coated particlesin all 3 cases.

Example 7

A bone marrow suspension obtained from a breast cancer patient wasstudied to examine whether micrometastic tumor cells could be present.After the preparation of mononuclear cells, these were incubated withthe same 3 anti-carcinoma antibodies used in the example above, but isthis case the antibodies were first attached to Dynabeads SAM IgGparamagneteic particles. After 1 incubation with these directly coatedparticles, the cell suspension was examined in the microscope, and ahigh number of cells were found positive with a number ofparticle-rosettes attached to their membrane.

Similar experiments have been performed in a number of pleural orascitic effusion and bone marrow from patients with breast cancer.

Example 8

T47D human breast carcinoma cells were incubated for varying lenghts oftime with Hoechst fluoresence dye, and the viability of the labeledcells was checked. Varying numbers of labeled breast carcinoma cellswere then added to 1×10⁶ bone marrow cells obtained from healthyvolunteers. In different experiments, different concentrations ofparamagnetic, monodisperse particles (Dynabeaos P450) coated withindividual anticarcinoma antibodies (NrLu10, MOC31, or 12H12) wereadded. After incubation for 30 min on ice, samples of the different testtubes were examined in a counting chamber under light and fluorescencemicroscopy. When the ratio of tumor cells/total nucleated cells was low,the cell suspension was subjected to a magnetic field and the cells withparticles attached were isolated before examined in the microscope. Itwas found that at an optimal ratio of 1-10 paramagnetic beads per tumorcell in the cell mixture, all the tumor cells had from 2-15 beadsattached to their surface. The sensitivity of the detection method wasclose to one target-cell per 10⁴ nucleated cells. In control experimentswith labeled tumor cells using antibodies known to have somecross-reativity to normal cells, this cross-reactivity was confirmedwith the antibody-coated paramagnetic particles. In experiments withbeads without tumor-associated antibody coating, none of the targetcells bound any beads.

Similar experiments have been performed both with other breast cancerlines and a small cell lung cancer cell line. Similar sensitivity andspecificity were obtained in these experiments.

Example 9

Pleural and ascites fluid from patients with breast cancer and ovariancarcinoma were sentrifuged, the same coated paramagnetic particles usedin Example 1 were added, incubated and concentrated in a magnetic fieldbefore the suspension was examined under light microscopy. Typically,cells that had the clear morphological features of tumor cells had beadsattached, whereas none of the few normal cells bound the antibody-coatedbeads. In two cases with pleural effusion, an independent morphologicalexamination did not reveal the presence of any tumor cells, whereas asignificant number malignant cells were detected by the use ofantibody-coated beads. In some cases, tumor cells were separated in amagnetic field and transferred to tissue culture flasks containinggrowth medium specially prepared for growing breast cancer cells, inattempts to establish permanent cell lines from these cultures. Inparallel, cells from the malignant effusions were cultivated directlywithout positive selection with magnetic beads. In the latter cases, nocell line could be established, whereas in more than 50% of the caseswhere positively selected tumor cells had been used, cell lines weresuccessfully established.

Example 10

In some cases, bone marrow and peripheral blood obtained from patientswith breast cancer were examined with the present procedure by addingantibody-coated paramagnetic beads, incubating for 30 min at 4° C. andconcentrating in a magnetic field and by examining the suspension underlight microscopy. In both cases binding of the paramagnetic beads totumor cells, representing 0,1-1% of the nucleated cells in the bonemarrow and blood was detected, cells that could not be identified by anyother method.

Example 11

Antibodies against certain growth factor receptors or other geneproducts expressed on the surface of specific cell populations may beused to identify and positively select these cells. Beads coated withanti-transferrin receptor antibodies, used in the novel method accordingto the present invention were shown to represent a rapid, simple andsensitive method for identification of cells expressing thetransferrin-receptor.

Example 12

For various purposes isolation of specific populations of normal cellsis warranted. Endothelial cells lining the capillary or small vessels innormal or tumorous tissue could be positively selected from cellsuspensions prepared from the relevant tissues. The procedure involvedthe use of beads coated with antibody directed against structuresexpressed on the endothelial cells, but not on the other normal cells inthe cell mixture.

Example 13

Human cells injected into immunodeficient rodents was shown to bepresent in cell suspensions prepared from tumor xenografts and fromvarious host organs/tissues by employing magnetic particles coated withan anti-pan human antibody.

TABLE 1 MONOCLONAL ANTIGENS ANTIBODIES Adhesion molecules Fibronectinreceptor (α5β1 integrin) Pierce 36114, BTC 21/22 Calbiochem 341649Integrin α3 β1 M-Kiol 2 Vitronectin receptor (αvβ3 integrin) TP36.1, BTC41/42 Integrin α2 Calbiochem 407277 Integrin α3 Calbiochem 407278Integrin α4 Calbiochem 407279 Integrin α5 Calbiochem 407280 Integrin αVCalbiochem 407281 Integrin β2 Calbiochem 407283 Integrin β4 Calbiochem407284 GpIIβIIIα S231 ICAM-I (CD54) C57-60, CL203.4, RR1/1¹ VCAM-1Genzyme 2137-01 ELAM-1 Genzyme 2138-01 E-selectin BBA 8P-selectin/GMP-140 BTC 71/72 LFA-3 (CD58) TS 2/9 CD44 BM 1441 272, 25.32CD44-variants 11.24, 11.31, 11.10 N-CAM (CD56) MOC-1 H-CAM BCA9 L-CAM BM1441 892 N-CAM TURA-27 MACAM-1 NKI-M9 E-cadherin BTC 111, HECD-1, 6F9P-cadherin NCC-CAD-299 Tenascin BM 1452 193, Calbiochem 580664Thrombospondin receptor (CD36) BM 1441 264 VLA-2 A1.43 Laminin receptorHNK-1 epitope HNK-1 Carbohydrate antigens T-antigen HH8, HT-8 Tn-antigenTKH6, BaGs2 Siaiyl Tn TKH-2 Gastrointestinal cancer associated CA 19-9antigen (M_(w)200kD) Carcinoma associated antigen C-50 Le^(γ) MLuC1,BR96, BR64 di-Le^(r), tri-Le^(λ) B3 Dimeric Le^(λ) epitope NCC-ST-421H-type 2 B1 CA15-2 epitope CA15-3 CEA I-9, I-14, I-27, II-10, I-46,Calbiochem 250729 Galb1-4GlcNac (nL4, 6, 8) 1B2 H-II BE2 A type 3 HH8Lacto-N-fucopentanose III (CD15) PM-81 Glycolipids GD₃ ME 36.1, R24 GD₂ME36.1, 3F8, 14.18 Gb₃ 38-13 GM₃ M2590 GM₂ MKI-8, MKI-16 FucGM₁ 1D7, F12Growth factor receptors EGF receptor 425.3, 2.E9, 225 c-erbB-2 (HER2) BM1378 988, 800 E6 PDGFα receptor Genzyme 1264-00 PDGFβ receptor Sigma P7679 Transferrin receptor OKT 9, D65.30 NGF receptor BM 1198 637 IL-2receptor (CD25) BM 1295 802, BM 1361 937 c-kit BM 428 616, 14-A3,ID9.3D6 TNF-receptor GEnzyme 1995-01, PAL-M1 NGF receptor Melanomaantigens High molecular weight antigen 9.2.27, NrML5, 225.28, (HMW250.000) 763.74, TP41.2, IND1 Mw105 melanoma-associated glycoproteinME20 100 kDa antigen (melanoma/carcinoma) 376.96 gp 113 MUC 18 p95-100PAL-M2 Sp75 15.75 gr 100-107 NKI-bereb MAA K9.2 M_(r)125kD (gp125) Mab436 Sarcoma antigens TP-1 and TP-3 epitope TP-1, TP-3 M_(w)200kD 29-13,29.2 M_(w)160kD 35-16, 30-40 Carcinoma markers MOC-31 epitope (cluster 2epithelial MOC-31, NrLu10 antigen) MUC-1 antigens (such as DF3-epitopeMUC-1, DF3, BCP-7 to (gp290kD)) −10 MUC-2 and MUC-3 PMH1 LUBCRU-G7epitope (gp 230kD) LUBCRU-G7 Prostate specific antigen BM 1276 972Prostate cancer antigen E4-SF Protate high molecular antigen M_(w) > 400kD PD41 Polymorphic epithelial mucins BM-2, BM-7, 12-H-12 Prostatespecific membran antigen (Cyt-356) 7E11-C5 Human milk fat globulinImmunotech HMFG-1, 27.1 42kD breast carcinoma epitope B/9189 M_(w) > 10⁶mucin TAG-72, CC-49, CC-83 Ovarian carcinoma OC125 epitope OC125 (m_(w)750kD) Pancreatic HMW glycoprotein DU-PAN-2 Colon antigen Co17-1A(M_(w)37000) 17-1A G9-epitope (colon carcinoma) G9 Human colonicsulfomucin 91.9H M_(w)300kD pancreas antigen MUSE11 GA 733.2 GA733,KS1.4 TAG 72 B72.3, CC49, CC83 Undefined Oat1, SM1 Pancreaticcancer-associated MUSE 11 Pancarcinoma CC49 Prostateadenocarcinoma-antigen PD 41 M_(w)150-130kD adenocarcinoma of the lungAF-10 gp160 lung cancer antigen anti gp160 (Cancer Res. 48, 2768, 1988)M_(w)92kD bladder carcinoma antigen 3G2-C6 M_(w)600kD bladder carcinomaantigen C3 Bladder carcinoma antigen (Cancer Res. 49, AN43, BB369 6720,1989) CAR-3 epitop M_(w) > 400 kD AR-3 MAM-6 epitope (C15.3) 115D8 Highmolecular ovarian cancer antigen OVX1, OVX2 Mucin epitope Ia3 Ia3Hepatocellular carcinoma antigen M_(w)90kD KM-2 Hepernal epitope (gp43)Hepatocellular Hepema-1 carc. ag O-linked mucin containing 3E1.2N-glycolylneuraminic acid M_(w)48kD colorectal carcinoma antigen D612M_(w)71kD breast carcinoma antigen BCA 227 16.88 epitope (colorectalcarcinoma antigen) 16.88 CAK1 (ovarian cancers) K1 Colon specificantigen p Mu-1, Mu-2 Lung carcinoma antigen M_(w)350-420kD DF-L1, DF-L2gp54 bladder carcinoma antigen T16 gp85 bladder carcinoma antigen T43gp25 bladder carcinoma antigen T138 Neuroblastoma antigensNeuroblastoma-associated, such as UJ13A UJ13A epitope Glioma antigensMel-14 eptiope Mel-14 Head and neck cancer antigens M_(w)18-22kD antigenE48 HLA-antigens HLA Class 1 TP25.99 HLA-A VF19LL67 HLA-B H2-149.1HLA-A2 KS1 HLA-ABC W6.32 HLA-DR, DQ, DP Q 5/13, B 8.11.2β₂-microglobulin NAMB-1 Apoptosis receptor Apo-1 eptiope Apo 1 VariousPlasminogen activator antigens & receptors Rabbit polyclonalp-glycoprotein C219, MRK16, JSB-1, 265/F4 cathepsin D CIS-Diagnostici,Italy biliary epithelial antigen HEA 125 neuroglandular antigen (CD63)ME491, NKI-C3, LS62 CD9 TAPA-1, R2, SM23 pan-human cell antigen pan-H

1. A method for detecting a specific living target cell in a cellsuspension of a mixed cell population, in a fluid system containing amixed cell population, or in a cell suspension prepared from a solidtissue with the exception of normal and malignant hematopoietic cells inblood and bone marrow, the method comprising the steps of: a. coatingparamagnetic particles or beads with a fist antibody or antibodyfragment directed against a second antibody or antibody fragment; b.incubating the second antibody or antibody fragment with the cellsuspension to bind the second antibody or antibody fragment with thetarget cell, thereby creating a cell mixture, wherein the secondantibody or antibody fragment is directed against a membrane structurespecifically expressed on the target cell and not on a non-target cellin the cell mixture; c. washing the cell mixture to remove unboundsecond antibody or antibody fragment; d. mixing the coated paramagneticparticles or beads with the washed cell mixture; e. incubating thewashed cell mixture and the coated paramagnetic particles under gentlerotation at about 4° C. until target cell-bead rosettes are formed; andg. visually detecting the target cell-bead rosettes after incubation;wherein the target cells are living and can be detected at a sensitivityof one target cell per 100 or more total cells.
 2. The method of claim1, wherein the paramagnetic particle or bead is coated with a monoclonalmurine or a human antibody or fragment thereof.
 3. The method of claim1, wherein incubating lasts for 5-10 minutes to 2 hours.
 4. The methodof claim 3, wherein incubating lasts 30 minutes.
 5. The method of claim1, wherein the method further comprises the step of: pre-incubating theantibody-coated paramagnetic particle and the cell suspension with milddetergent.
 6. The method of claim 5, wherein the preincubating comprisesas detergent polyoxyethyleresorbitan monolaurate at a concentration lessthan 0.1% and the preincubation last 30 minutes at 4° C.
 7. The methodof claim 1, further comprising the steps of: isolating the targetcell-bead rosettes by applying a magnetic field to separate therosettes.
 8. The method of claim 1, wherein the second antibody orfragment thereof is directed against an antigen or a receptor in a cellwith abnormal developmental patterns.
 9. The method of claim 8, whereinthe cell is a primary or a metastatic cancer cell.
 10. The method ofclaim 8, wherein the second antibody or antibody fragment is directedagainst breast, ovarian or lung carcinoma cells; melanoma sarcoma,glioblastoma or cancer cells of the gastrointestinal tract; melanoma,sarcoma, glioblastoma or cancer cells of the genitourinary tract; ormelanoma, sarcoma, glioblastoma or cancer cells of thereticuloendothelial system.
 11. The method of claim 1, wherein themonoclonal antibody or fragment is of IgG isotype a F(ab′)₂ fragment, aF(ab) fragment, IgM, or a fragment of IgM.
 12. The method of claim 1,wherein the mixed cell population comprises mammalian tissue, a pleuraleffusion, a peritoneal effusion, a body fluid, or a solid tumor in anormal tissue or organ.
 13. The method of claim 12, wherein themammalian tissue comprises human bone marrow or human peripheral blood;tho body fluid comprises urine cerebrospinal fluid, semen, or lymph; orthe normal tissue or organ comprises liver, lymph node, spleen, lung,pancreas, bone, central nervous system, prostate gland, skin, or mucousmembranes.
 14. The method of claim 1, wherein the second antibody orantibody fragment is directed against fibronectin receptor, β-integrin,vitronectin receptor, αγβ3-integrin P-seletin including GMP-140,CD44-variants, N-CAM including CD-56, E-cadherin, Le^(γ),carcinoembryonic antigen or CEA, EGF receptor, c-erbB-2 including HER2,transferin receptor, TNF-receptor, high molecular weight antigen,p95-100, sarcoma antigens including TP-1 and TP-3 epitope, Mv 200 kD,Mv160 kD, MOC-31 epitope including cluster 2 epithelial antigen, MUC-1antigen including DF3-epitope and gp290 kD, prostate high molecularantigen, TAG 72, bladder carcinoma antigen, Mv 48 kD colorectalcarcinoma antigen, lung carcinoma antigen Mv 350-420 kD, Me1-14 epitope,β₂-microglobulin, Apo-1 epitope, or pan-human cell antigen.
 15. Themethod of claim 1, wherein the second antibody or antibody fragment isdirected against a growth factor receptor or an oncogene productexpressed on the membrane of a malignant cell.
 16. The method of claim1, wherein the second antibody or antibody fragment directed against amembrane structure specifically expressed on the target-cell is a murineor a human antibody or fragment thereof.
 17. The method of claim 1,wherein the method further comprises after incubating, applying amagnetic field to separate out the target cell-bead rosettes.
 18. Themethod of claim 1, wherein visually detecting includes counting thetarget cell-bead rosettes using a microscope or a cell or particlecounting device.
 19. The method according to claim 1, further comprisingafter incubating; applying a magnetic field to the mixture to separateout the target cell-bead rosettes; and detecting target cell specificgenes.
 20. The method according to claim 1, wherein the first antibodyor antibody fragment is a monoclonal antibody or antibody fragment, thesecond antibody or antibody fragment is a monoclonal antibody orantibody fragment, or the first and second antibodies or antibodyfragments are monoclonal antibodies or antibody fragments.
 21. Themethod according to claim 1, wherein the visually detecting includesconjugating a detectable label to the second antibody.
 22. The methodaccording to claim 1, wherein the target cells are detected at asensitivity of one target cell per 1000 or more total cells.
 23. Themethod according to claim 1, wherein the second antibody is an IgGantibody and the first antibody recognizes the Fc-portion of the secondantibody.
 24. A kit for performing the method of claim 1, the kitcomprising: a. a first antibody, where said first body is a specificmonoclonal antibody or antibody fragment directed against a secondantibody or antibody fragment, said first antibody coating aparamagnetic particle or bead without removing its antigen-bindingability; b. a paramagnetic particle or bead; and c. the second antibody,wherein said second antibody is a specific monoclonal antibody orantibody fragment directed against antigen or a receptor within or onthe target cell; wherein said second antibody or antibody fragment isconjugated to a detectable label.
 25. The kit of claim 24, wherein thedetectable label is an enzyme peroxidase or alkaline phosphatase. 26.The kit of claim 24, comprising a paramagnetic particle or bead coatedwith the first antibody and a paramagnetic particle or bead not coatedwith antibody.
 27. A kit for performing the method of claim 1, the kitcomprising: a. a first antibody, wherein said first antibody is aspecific monoclonal antibody or antibody fragment directed against asecond antibody or antibody fragment, said first antibody coating aparamagnetic particle or bead without removing its antigen-bindingability; b. a paramagnetic particle or bead; and c. the second antibody,wherein said second antibody is a specific monoclonal antibody orantibody fragment directed against an antigen or a receptor within or onthe target cell, wherein the second antibody or antibody fragment isdirected against fibronectin receptor, β-integrin, vitronectin receptor,αγβ3-integrin. P-seletin including GMP-140, CD44-variants, N-CAMincluding CD-56, E-cadherin, Le^(γ), carcinoembryonic antigen or CEA,EGF receptor, c-erbB-2 including HER2, transferin receptor,TNF-receptor, high molecular weight antigen, p95-100, sarcoma antigensincluding TP-1 and TP-3 epitope, Mv 200 kD, Mv160 kD, MOC-31 epitopeincluding cluster 2 epithelial antigen, MUC-1 antigen includingDF3-epitope and gp 290 kD, prostate high molecular antigen, TAG 72,bladder carcinoma antigen, Mv 48 kD colorectal carcinoma antigen, lungcarcinoma antigen Mv 350-420 kD, Me1-14 epitope, γ₂-microglobulin, Apo-1epitope, or pan-human cell antigen; wherein said second antibody orantibody fragment is conjugated to a detectable label.
 28. A method fordetecting living tumor cells in a cell suspension of mixed cellpopulation or in a cell suspension prepared from a solid tissue, withthe exception of normal and malignant hematopoietic cells in blood andbone marrow, comprising: a) coating paramagnetic particles with a firstantibody or fragment directed against a second tumor-specific monoclonalantibody or fragment; b) incubating the second tumor specific antibodyor antibody fragment with the cell suspension to allow the second tumorspecific antibody or antibody fragment to bind the tumor cells; c)washing the cell suspension to remove unbound second antibody orantibody fragment; d) mixing the coated paramagnetic particles with thecell suspension; e) incubating the mix at about 4° C. under gentlerotation until tumor cell-bead rosettes are formed; and f) visuallydetecting the tumor cell-bead rosettes; wherein the target cells areliving and can be detected at a sensitivity of one target cell per 100or more total cells.
 29. The method according to claim 28, furthercomprising after incubating; applying a magnetic field to the mixture toseparate out the tumor cell-bead rosettes.
 30. The method according toclaim 28, wherein the tumor-specific monoclonal antibody is specific fortumor antigens comprising a growth factor receptor, an oncogene productexpressed on the membrane of a malignant cell, an adhesion membranemolecule, an MDR protein, breast, ovarian or lung carcinoma cells;melanoma, sarcoma, glioblastoma or cancer cells of the gastrointestinaltract; melanoma, sarcoma, glioblastoma or cancer cells of thegenitourinary tract; or melanoma, sarcoma, glioblastoma or cancer cellsof the reticuloendothelial system.
 31. The method according to claim 28,wherein the mixture is incubated for about 30 minutes.
 32. The methodaccording to claim 28, wherein the target cells are detected at asensitivity of one target cell per 1000 or more total cells.